The invention relates generally to a dietary supplement that increases DNA methylation, methods for altering the phenotype of offspring using the supplement, and methods for inhibiting retroviral expression using the supplement. The invention also relates to an animal model for epigenetic regulation of phenotypic expression.
The maternal reproductive tract, arguably, is the environment most critical to the developing mammalian embryo. Its metabolic and physiologic characteristics modulate the zygote""s development through all embryonic stages until birth. Indeed, the conditions in the embryo""s immediate milieu seem to determine many characteristics and susceptibilities of the adult organism.
Mammalian development is dependent on DNA methyltransferase (MTase) and its product 5-methylcytosine (5MC) to help establish, define, or stabilize the various cell types that constitute the developing embryo. In mammals, 5MC is a major epigenetic mechanism, with some 5MC patterns being inherited epigenetically. DNA MTase requires S-adenosylmethionine (SAM) and uses zinc as a cofactor.
Synthesis of the chief methyl donor SAM is dependent on dietary folates, vitamin B12, methionine, choline, and betaine, which are also available as nutritional methyl supplements. In the human maternal diet, folic acid is important for the prevention of neural tube birth defects, where it may act via methyl metabolism although the mechanism is presently unknown. Little is known about how, or if, maternal dietary methyl supplements affect epigenetic regulation of the developing mammalian embryo or whether high levels of certain methyl supplements are toxic. Cooney proposed that dietary methyl supplements given to adults could affect gene expression and 5MC levels in adults and that the level of gene-specific 5MC in young mammals could affect their adult health and longevity (Cooney, C. A. 1993 Growth Dev. Aging 57, 261-273).
The mouse agouti alleles, Aw and A, regulate the alternative production of black (eumelanin) and yellow (pheomelanin) pigment in individual hair follicles. Transcription of the gene occurs only in the skin during the short period when the yellow subapical band is formed at the beginning of each hair growth cycle. This cyclic expression results in the xe2x80x98agoutixe2x80x99 coat pattern.
Due to mutations in the regulatory region of the agouti locus, mice bearing the dominant xe2x80x98viable yellowxe2x80x99 (Avy), xe2x80x98IAPyellowxe2x80x99 (Aiapy), or xe2x80x98hypervariable yellowxe2x80x99 (Ahvy) alleles synthesize much more pheomelanin than eumelanin. These mutations arose through spontaneous insertions of single intracisternal A particle (IAP) sequences in different regions of the agouti gene, all preceding the first coding exon. In these yellow mice, the gene is under control of the IAP promoter/enhancer and is therefore transcribed continuously in essentially all tissues. This results not only in yellow hair but also in obesity, hyperinsulinemia, diabetes, increased somatic growth, and increased susceptibility to hyperplasia and tumorigenesis.
IAP""s are retrovirus-like particles produced from the expression of a repetitive DNA element endogenous in mammalian genomes. The term is also used for the DNA i.e. the endogenous retrovirus-like DNA sequence (repetitive DNA element). IAP expression is controlled by DNA methylation (i.e. 5MC). More specifically the long terminal repeat (LTR) of many retroviruses and retrovirus-like elements like the IAP are controlled by DNA methylation. DNA methylation prevents LTRs from activating and driving gene expression. Many LTRs and IAPs are constitutively active unless methylated. Expression of IAPs and other retrovirus and retrovirus-like elements results in a variety of health problems in mice, humans, and other mammals.
Mice of the non-agouti genotype, a/a, are black in coat color and no pheomelanin is synthesized, except in hair follicles in the ears and perineal area, due to insertions of non-IAP retroviral sequences in the intron preceding the first coding exon of the agouti gene. Because the a allele produces neither yellow nor pseudoagouti phenotypes, it is often used as the second allele in studies with the dominant yellow mutants.
Expression of Avy, Aiapy, and Ahvy can be down-regulated epigenetically. In yellow Aiapy/a and Ahvy/a mice, the proximal IAP long terminal repeat (i.e. IAP LTR), containing promoters and enhancers, is hypomethylated, whereas in pseudoagouti Aiapy/a mice and their black Ahvy/a homologs, the LTR is methylated. Thus, in pseudoagouti mice the IAP promoter is inactive, allowing the normal agouti promoters to regulate transcription of the gene. The mouse genotypes Avy/a and Aiapy/a are expressed in almost identical phenotypes. Since the agouti protein is continuously and ectopically expressed in both mutants, it is likely that in Avy/a mice, a regulatory mechanism is operative similar to that reported for Aiapy and Ahvy.
A continuous spectrum of variegated patterns of eumelanic mottling (EM), i.e., agouti, on a yellow background characterizes Avy/a mice. Their phenotypes are defined by the degree of EM (see the list of phenotypes in Example 1). Thus, a xe2x80x98clear yellowxe2x80x99 mouse (all yellow) is at one extreme of the EM spectrum and the xe2x80x98pseudoagoutixe2x80x99 mouse (all agouti, FIG. 1) occupies the other extreme. The latter resembles the agouti (A/xe2x88x92) coat color phenotype, does not become obese, is normoinsulinemic, and is less susceptible to tumorigenesis.
Epigenetic changes are changes in the use or expression of genes and other DNA sequences by processes that do not change the DNA coding sequence itself. Epigenetic processes result in changes that are heritable from one cell generation to the next, thus providing for the maintenance of cellular differentiation. Epigenetic changes and processes can be heritable from one animal generation to the next. Epigenetic processes allow us to go from a single cell (fertilized egg or zygote) which is one cell type at conception, to a multicellular organism with many cell types as we develop as embryos and beyond. Epigenetic processes allow us to maintain our established cell types whether we are embryos, infants, children or adults. It is the breakdown of these epigenetic processes that contribute to the development of cancer as well as to aging and likely a number of other diseases. Most of these same epigenetic processes occur in all plants and animals studied. In particular fish, birds, mammals, plants, reptiles, amphibia and some fungi use DNA methylation as an epigenetic DNA and gene control mechanism. Importantly all mammals studied appear to use the same set of epigenetic mechanisms.
All mammals studied, especially all placental mammals, including humans and domesticated mammals, use basically the same epigenetic control mechanisms including DNA methylation, methylated DNA binding proteins, differential DNA replication timing in the cell cycle, histone acetylation, and differential chromatin condensation, to name a few common features. All mammals studied, especially all placental mammals, including humans and domesticated mammals, use basically the same methyl metabolism, have basically the same dietary requirements for methyl metabolism (albeit met by various means), have DNA MTase enzymes which require S-adenosylmethionine and are inhibited by S-adenosylhomocysteine and use zinc as a cofactor, as well as having numerous other features in common. These common features make it reasonable to expect that effects on basic processes in all mammals, especially all placental mammals, including humans and domesticated mammals, would share common features. Thus it would be reasonable to expect that specific dietary supplements or specifically altered dietary balances would affect the epigenetics and/or phenotype of offspring in all mammals, especially all placental mammals, including humans and domesticated mammals.
Many of the mechanisms used to control gene, repetitive sequence, repetitive DNA element, retrovirus, transposon and intragenomic parasite expression are common to a very broad range of organisms. For example birds, reptiles, fish, other vertebrates, some fungi, and most plants studied use DNA methylation as a gene, repetitive sequence, repetitive DNA element, retrovirus, transposon and intragenomic parasite control mechanism. Likewise these and other eukaryotes have in common with mammals certain other epigenetic and gene, repetitive sequence, repetitive DNA element, retrovirus, transposon and intragenomic parasite control mechanisms. Several determinants of epigenetic inheritance are outlined in Example 6.
Morphological changes are not necessarily epigenetic in nature or in source. Therefore a change such as described by Meck et al. 1988 (Dev. Psychobiol. 21, 339-353) where maternal choline supplementation affects the memory of offspring is not known to be epigenetic, not known to involve epigenetic mechanisms and no specific gene or even DNA is known to be involved.
In Avy/a mice, there is partial maternal epigenetic inheritance of phenotype. In general, maternal epigenetic inheritance occurs when the epigenetic phenotype and/or allelic expression of the mother is a determinant of the epigenetic phenotype and/or allelic expression of the offspring. For Avy/a dams, the proportion of pseudoagouti offspring depends on the mother""s agouti locus epigenetic phenotype.
In both Avy/a and Aiapy/a mice, the proportions of zygotes differentiating into pseudoagouti phenotype offspring are determined by the gender of the parent contributing the mutant allele, as well as by the dam""s strain genome. These gender and strain effects demonstrate, respectively, genomic imprinting and strain-specific modification of the Avy and Aiapy alleles.
Genomic imprinting occurs when the level of allelic expression in offspring depends on the gender of the parent contributing the allele. Imprinting is a parental gender effect on gene expression in offspring but is neither the inheritance of a parent""s epigenetic somatic characteristics nor the inheritance of a parent""s somatic allelic imprint (although these may happen to coincide between parent and offspring).
One aspect of the invention provides a maternal nutritional supplement which can positively affect health and longevity of the offspring comprising at least two of the following: about 5-15 g/kg diet/day of Choline, about 5-15 g/kg diet/day of Betaine, about 5-15 mg/kg diet/day of Folic acid, about 0.5-1.5 mg/kg diet/day of Vitamin B12, about 0 to 7.5 g/kg diet/day of L-Methionine, and 0 to 150 mg/kg diet/day of Zinc. The concentrations are preferably about 10-15 g/kg diet/day of Choline, about 10-15 g/kg diet/day of Betaine, about 10-15 mg/kg diet/day of Folic acid, about 1.0-1.5 mg/kg diet/day of Vitamin B12, about 5 to 7.5 g/kg diet/day of L-Methionine, and 100 to 150 mg/kg diet/day of Zinc. The concentrations are even more preferably about 15 g/kg diet/day of Choline, about 15 g/kg diet/day of Betaine, about 15 mg/kg diet/day of Folic acid, about 1.5 mg/kg diet/day of Vitamin B12, about 7.5 g/kg diet/day of L-Methionine, and 150 mg/kg diet/day of Zinc.
The expression xe2x80x9ckg/diet/dayxe2x80x9d or xe2x80x9cper kg diet per dayxe2x80x9d means daily or equivalent, regular, supplementation at a particular level per kilogram of food or diet that would normally be eaten. Supplementation may be taken with, at the same time as, or at a different time than, the food or diet. Supplementation could be taken on a day, or on days, when food isn""t eaten or when food or diet is restricted but where the supplementation level is approximately equivalent to that which would be taken in a day if food or diet were consumed at a xe2x80x9cnormalxe2x80x9d ad libitum rate and if food or diet contained the supplement at the particular level per kilogram of food or diet.
A further aspect of the invention provides a method for screening for substances that affect epigenetic changes in offspring. The method uses a model consisting of a recessive non-agouti (a/a) female mouse which has been or will be mated with a heterozygous viable yellow male mouse (Avy/a). The substance or treatment is administered to the unborn offspring and the phenotype of the offspring is measured after birth. In one embodiment the substance can be administered by administering it to the mother before and/or during pregnancy. In another embodiment it can be administered in vivo. In another embodiment it can be administered in vitro to a fertilized egg, zygote, embryo or fetus. In one embodiment the measurement can be by visual examination of the coat color pattern. In another embodiment the measurement can be by quantitation of 5-methylcytosine in a cell, tissue or body fluid. In a further embodiment the measurement can be by quantitation of DNA methylation of the IAP LTR. An additional way to express use of these treatments is to prepare or condition the female and her oocytes in vivo or in vitro in order to affect the developmental processes following fertilization.
A further aspect of the invention is a method for screening for substances that affect the expression of parasitic DNA sequences. The method uses a model consisting of a pregnant recessive non-agouti (a/a) female mouse which has been mated with a heterozygous viable yellow male mouse (Avy/a). The substance or treatment is administered to the unborn offspring and the phenotype of the offspring is measured after birth. In one embodiment the substance can be administered by administering it to the mother before and/or during pregnancy. In another embodiment it can be administered in vivo. In another embodiment it can be administered in vitro to a fertilized egg, zygote, embryo, or fetus. In one embodiment the measurement can be by visual examination of the coat color pattern. In another embodiment the measurement can be by quantitation of 5-methylcytosine in a cell, tissue or body fluid. In a further embodiment the measurement can be by quantitation of DNA methylation of the IAP LTR. In a further embodiment the parasitic DNA sequences are those for which expression is regulated by DNA methylation.
A further embodiment of the invention is a method for changing the epigenetically determined phenotype and inhibiting parasitic DNA sequences by administering the pharmaceutical of Claim 27 to an offspring before birth. In one embodiment the substance can be administered by administering it to the mother before and/or during pregnancy. In another embodiment it can be administered in vivo. In another embodiment it can be administered in vitro to a fertilized egg, zygote, embryo or fetus. An additional way to express use of these treatments is to prepare or condition the female and her oocytes in vivo or in vitro in order to affect the developmental processes following fertilization.
Yet another embodiment of the invention is a method for inhibiting expression of an IAP sequence in an unborn offspring by administering the pharmaceutical of Claim 27 to an offspring before birth. In one embodiment the substance can be administered by administering it to the mother before and/or during pregnancy. In another embodiment it can be administered in vivo. In another embodiment it can be administered in vitro to a fertilized egg, zygote, embryo or fetus. An additional way to express use of these treatments is to prepare or condition the female and her oocytes in vivo or in vitro in order to affect the developmental processes following fertilization.
Still another embodiment of the invention is a method for reducing susceptibility to tumor-formation, and improving the health and longevity of an unborn offspring by administering the pharmaceutical of Claim 27 to an offspring before birth. In one embodiment the substance can be administered by administering it to the mother before and/or during pregnancy. In another embodiment it can be administered in vivo. In another embodiment it can be administered in vitro to a fertilized egg, zygote, embryo, or fetus. An additional way to express use of these treatments is to prepare or condition the female and her oocytes in vivo or in vitro in order to affect the developmental processes following fertilization.